Project Summary A critical question in the field of Epigenetics/Mammalian Gene Regulation is how a cellular identity is inherited by progeny cells during cell division. This fundamental aspect of epigenetic regulation was recently clarified in our lab: repressed, but not active, chromatin domains are inherited. Repressed chromatin domains in facultative heterochromatin are maintained by the multi-subunit complex, Polycomb Repressive Complex 2 (PRC2), that catalyzes the histone post-translational modification, H3K27me3. PRC2 exhibits a notable ?read and write? feature whereby its recognition of H3K27me3 results in its allosteric activation. Thus, PRC2 can fully restore repressive chromatin domains upon inheritance of nucleosomes comprising H3K27me3. Remarkably, our findings point to a previously reported histone chaperone, NPM1, as facilitating this inheritance of repressed chromatin: NPM1 is exclusively localized to chromatin in late S-phase when repressed chromatin is replicated, and NPM1 interacts directly with PRC2. Notably, the NPM1 mutant (NPM1c) associated with ~35% of all Acute Myelogenous Leukemia (AML) is mis-localized to the cytoplasm. We will expand our mechanistic studies of epigenetic inheritance by investigating the role of NPM1 as an S-phase-specific histone chaperone and its interplay with PRC2 in vivo. We will further investigate the role of NPM1 in epigenetic inheritance by adapting our in vivo assay for chromatin domain inheritance as a function of the presence of NPM1 and pertinent NPM1 mutants. With a different perspective, we will follow the interactive dynamics of NPM1 and PRC2 in the context of a replication fork using single-molecule localization microscopy as well as stochastic optical reconstruction microscopy (STORM). These approaches should bear directly on the role of NPM1 as an ?epigenetic histone chaperone? and the significance of its interaction with the epigenetic regulator, PRC2. Importantly, we propose that the NPM1c mutant associated with AML results in a mis-regulated PRC2. Indeed, known HOX gene targets of PRC2 are aberrantly expressed in NPM1c AML, and participate in the establishment of the leukemic state. While deposition of H3K27me3 by PRC2 and DNA methylation by DNMT3A result in repressed chromatin, these events are usually mutually exclusive. Yet, NPM1 and DNMT3A mutations synergize in leukemogenesis. Thus, we further propose that DNMT3A partially compensates for our proposed NPM1c-mediated thwarting of PRC2?a compensation that is lost upon DNMT3A mutation. We are in position to fully grasp the sequence of aberrant epigenetic events as they occur during leukemogeneis. We will track the repercussions to gene expression, features of repressed chromatin domains, PRC2 chromatin occupancy and DNA methylation in both tissue culture and a mouse model of AML, as NPM1c is temporally expressed as a function of the presence of mutant DNMT3A. These results will pinpoint the initial, aberrant epigenetic events giving rise to AML. During the Bridging funding, we will concentrate on the in vivo cell based assay that we developed to precisely define the function of NPM1 during mitotic inheritance of H3K27me2/me3-repressive chromatin domains, as described in the Experimental section, below.